Quantitative Imaging of Single UpconversionNanoparticles in Biological TissueAnnemarie Nadort1,2, Varun K. A. Sreenivasan1, Zhen Song1, Ekaterina A. Grebenik1,3,
Andrei V. Nechaev4, Vladimir A. Semchishen5, Vladislav Y. Panchenko5, Andrei V. Zvyagin1,5*
1 MQ Biofocus Research Centre, Macquarie University, Sydney, NSW, Australia, 2 Department of Biomedical Engineering and Physics, Academic Medical Center, University
of Amsterdam, Amsterdam, The Netherlands, 3 Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia, 4 HTBAS
Department, Lomonosov Moscow State University of Fine Chemical Technologies, Moscow, Russia, 5 Institute of Laser and Information Technology, Russian Academy of
Sciences, Troitsk, Moscow Region, Russia
Abstract
The unique luminescent properties of new-generation synthetic nanomaterials, upconversion nanoparticles (UCNPs),enabled high-contrast optical biomedical imaging by suppressing the crowded background of biological tissueautofluorescence and evading high tissue absorption. This raised high expectations on the UCNP utilities for intracellularand deep tissue imaging, such as whole animal imaging. At the same time, the critical nonlinear dependence of the UCNPluminescence on the excitation intensity results in dramatic signal reduction at (,1 cm) depth in biological tissue. Here, wereport on the experimental and theoretical investigation of this trade-off aiming at the identification of optimal applicationniches of UCNPs e.g. biological liquids and subsurface tissue layers. As an example of such applications, we report on singleUCNP imaging through a layer of hemolyzed blood. To extend this result towards in vivo applications, we quantified theoptical properties of single UCNPs and theoretically analyzed the prospects of single-particle detectability in live scatteringand absorbing bio-tissue using a human skin model. The model predicts that a single 70-nm UCNP would be detectable atskin depths up to 400 mm, unlike a hardly detectable single fluorescent (fluorescein) dye molecule. UCNP-assisted imagingin the ballistic regime thus allows for excellent applications niches, where high sensitivity is the key requirement.
Citation: Nadort A, Sreenivasan VKA, Song Z, Grebenik EA, Nechaev AV, et al. (2013) Quantitative Imaging of Single Upconversion Nanoparticles in BiologicalTissue. PLoS ONE 8(5): e63292. doi:10.1371/journal.pone.0063292
Editor: Sangaru Shiv Shankar, King Abdullah University of Science and Technology, Saudi Arabia
Received December 11, 2012; Accepted March 29, 2013; Published May 14, 2013
Copyright: � 2013 Nadort et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors thankfully acknowledge the financial support by the Russian Foundation of Basic Research #11-04-12113, Russia, the Prins BernhardCultuur fonds (The Netherlands), and PGRF Macquarie University, Australia. The funders had no role in study design, data collection and analysis, decision topublish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: [email protected]
Introduction
Optical imaging of biological tissues provides highly informa-
tive, non-invasive and inexpensive means to assess the tissue
physiological status and functionality, especially for diagnosis of
pathological sites. Labeling these tissue sites with luminescent
biocomplexes, often referred to as molecular probes, improves
localization accuracy and sensitivity. Deployment of a new class of
molecular probes whose excitation/emission falls into the so-called
biological tissue transparency window (650 nm –1300 nm) allows
deeper imaging in virtue of the minimized absorption and
scattering of biotissue in this near-infra-red (NIR) wavelength
range [1,2]. For example, specific labeling of cancerous lesions
with a molecular probe based on indocyanine green (the only NIR
organic dye approved for routine clinical procedures) provided up
to a 4-fold increase in the tumor imaging contrast in comparison
with that achieved using the intrinsic hemoglobin spectral
signature and tissue scattering [3,4].
The development of NIR-emitting probes has traditionally been
directed towards organic dyes, but more recent studies have
demonstrated considerable promise of inorganic nanoparticle-
based probes, such as quantum dots (QDs) and most novel
upconversion nanoparticles (UCNPs) [5,6]. Organic NIR-dyes are
favorably small, easily dispersed in an aqueous environment and
are amenable to bioconjugation utilizing established protocols.
However, their poor thermal and photochemical stability (photo-
bleaching) and a low fluorescence quantum yield (QY) (5–25% in
NIR, with propensity to deteriorate in biological environments)
are inferior in comparison with QDs whose attractive optical
properties include size-tunable optical absorption and emission
spectra, high photochemical stability, a large QY (20–70% in
NIR), and high thermal stability [7]. On the other hand, QD
fluorescence is intermittent (‘blinking’) hindering applications such
as single molecule tracking, while QD intrinsic toxicity largely
precludes their use in vivo [8]. UCNPs share the high photochem-
ical and thermal stability of QDs and other inorganic nanomater-
ials, complemented with non-blinking emission and demonstrated
biocompatibility. In addition, UCNPs can be employed as a
docking platform for high drug payloads for targeted delivery [9].
The key advantage of UCNPs is their unique photochemical
structure that enables ‘‘upconversion’’ of NIR excitation light
(980 nm) of modest intensity (100 W/cm2) to the higher energy
visible emission (450–850 nm) [10]. Since no known biological
molecule is capable of such conversion [1,11], the intrinsic tissue
fluorescence, termed autofluorescence, can be eliminated in the
detection path by conventional optical short-pass filtering. In
addition, the exceptionally long (sub-ms) luminescence lifetimes of
PLOS ONE | www.plosone.org 1 May 2013 | Volume 8 | Issue 5 | e63292
UCNPs allow realization of time-gated detection schemes that can
completely suppress the residual back-scattered excitation light
bleeding through the (interference) spectral filters [12]. Since
in vivo imaging performance is crucially dependent on the contrast
provided by the molecular probe [1], background-free detection of
UCNPs is very promising, as has been shown by the autofluores-
cence-free trans-illumination imaging in mice using biocompatible
UCNPs [13,14].
The unique upconversion property of UCNPs is a result of the
sequential photon absorption and energy transfer processes within
an inorganic host matrix, with hexagonal-phase b-NaYF4 co-
doped with trivalent lanthanide ions reported to be the most
efficient [15,16]. These dopant ions are classified as sensitizers and
activators considering their respective roles in the UCNP
absorption and emission. The sensitizer, typically Ytterbium ion
(Yb3+), absorbs the NIR-radiation energy and transfers it non-
radiatively to the closely spaced neighboring Yb3+, forming a
network of excited Yb3+ (referred to as delocalized quasi-exciton
[17]) until the energy is seized by activator ions, usually Erbium
(Er3+), or Thulium (Tm3+). The activator makes a transition to a
metastable excited state, from where it can coalesce with a nearby
excited Yb3+-ion, to be transferred to the next energy level (a
process called energy transfer upconversion) at the expense of the
participating Yb3+ decaying to the ground state. Multiple-step
energy transfer upconversion is also possible. The activator can
return to the ground state by radiating a photon in the visible or
NIR wavelength range. The upconversion emission results from
the absorption of 2 or more NIR photons, and hence exhibits a
supralinear dependence on the excitation intensity, Iex, as
quantified by the conversion efficiency (guc) addressed in Section
2.1.2. In contrast with other anti-Stokes processes occurring at Iex
, 16105 W/cm2 [18], the energy transfer upconversion occurs
via a real metastable excited state(s) at rather modest Iex ,16102 W/cm2 that is readily achievable by focusing a continuous-
wave excitation beam.
The specific photophysical properties of upconversion nano-
particles entail several challenges for optical biomedical imaging.
Firstly, the quasi-excitonic nature of UCNP excitation renders the
emission size dependent, susceptible to surface quenching [19] and
vulnerable to (biological) environment, which causes reduction of
guc values that are generally small compared to these of QDs or
organic fluorescent dyes. Values of guc are typically around 1% for
nanoparticle diameters of ,50 nm, shown to be the optimal size
for cellular receptor-mediated internalization [20]. Secondly, guc is
dependent on the excitation intensity and increases to a plateau
value, for which the Iex is referred to as saturation intensity, Isat (Isat
%16102 W/cm2). Keeping Iex close to Isat is preferable for
biomedical imaging.
This dependency of guc on Iex poses the main challenge for
UCNP-assisted optical imaging in deep (live) tissue layers
exceeding centimeter(s). Indeed, the attainment of Isat by focusing
is hardly possible in turbid biotissue, while the laser power is
limited by the allowed maximum permissible exposures (MPEs).
The UCNP luminescence signal is therefore diminished with
depth as was demonstrated in tissue phantom experiments [14]. In
another recent study, the critical dependence of guc versus depth
was manifested by the considerable deterioration of axial and
lateral resolution of full-field upconversion microscopy at depths of
,300–400 mm [21]. These results urge identification of practical
application niches for UCNPs that will benefit from the afore-
mentioned advantages of UCNPs, while not being restricted by
their limitations. Promising scenarios include UCNP-imaging in
biological fluids, thick slices and subsurface tissue layers, where the
UCNP based molecular probe contrast is expected to be superior
to that of existing molecular probes, including organic fluorescent
dyes.
This work aims to explore the cutting-edge optical imaging
scenario represented by a single UCNP buried in an absorbing
biological environment, which was demonstrated by single-UCNP
imaging through a layer of hemolyzed blood. We believe this result
has not been reported before [22–25]. We report on absolute
conversion efficiency and spectral properties of single UCNPs. The
subsequent thorough characterization of the UCNP emission
signal versus the excitation/detection parameters allowed projec-
tion of the obtained experimental results onto a theoretical model,
generalized to biomedical imaging applications of extreme
sensitivity in challenging in vivo environments. Single-UCNP
imaging in live skin was modeled to be feasible in skin at depths
up to a few hundred micrometers, with superior contrast
compared to a conventional fluorescent dye molecule. The results
show that imaging in the ballistic regime allows for excellent
applications of UCNP-guided imaging in life sciences where high
sensitivity is a key requirement.
Materials and Methods
2.1 SynthesisSynthesis reagents. Y2O3, Yb2O3, Er2O3, sodium trifluor-
oacetate 99%, trifluoroacetic acid 98%, oleic acid 90%, 1-
octadecene 90% (all purchased from Sigma-Aldrich).
Synthesis protocol of b-NaYF4:Yb,Er nanophosphors. A
mixture of Y2O3 (0.78 mmol), Yb2O3 (0.20 mmol), Er2O3
(0.02 mmol) was suspended in 70% trifluoroacetic acid (20 ml)
and gently refluxed until a clear solution was obtained (< 6 h),
then cooled to room temperature. After the solution was
evaporated, the formed residue was dried in vacuum (0.1 torr,
3 h). The resulting slurry was thoroughly ground in agate mortar
to obtain a fine homogenous powder. This rare-earth trifluor-
oacetate mixture, together with sodium trifluoroacetate (1 mmol),
was added to oleic acid (6 ml) and 1-octadecene (6 ml) in a three-
neck flask equipped with a thermometer, septum stopper and glass
magnetic stirrer. Next, the solution was heated to 100uC and
stirred under vacuum for 30 min for degassing and removal of
water. The mixture was subsequently gradually heated at a rate of
8uC/min to 290uC, and maintained at this temperature for
45 min under argon atmosphere. Next, a solution of sodium
trifluoroacetate (1 mmol) in oleic acid (2 ml) and of 1-octadecene
(2 ml), heated to 85uC, was added to the reaction, after which the
reaction temperature was raised to 330uC and stirred for 15 min
under argon. Next, isopropanol (130 ml) was added to the cooled
solution and the mixture was centrifuged at 6000 rpm for 30 min.
The resultant nanoparticles were washed with absolute ethanol (4
times), dried, dissolved in chloroform (10 ml), precipitated with
isopropanol (50 ml) and centrifuged at 4000 rpm for 10 min. The
last procedure was repeated 2 times. The residue was dried at
room temperature.
2.2 Imaging2.2.1 Transmission electron microscopy. The UCNPs
were dissolved in hexane, drop-casted on Formav�-coated TEM
grids and dried in a desiccator at room temperature. The grids
were imaged with a Philips CM10 TEM and analyzed using
ImageJ free-ware to obtain the UCNP size distributions. For
single-UCNP imaging, the hexane solutions were diluted and
drop-casted on Formav�-coated TEM nickel finder grids for easy
navigation. The thinly coated finder grids can be imaged using
TEM and optical epi-luminescence systems. In order to find single
nanoparticles, low-magnification TEM imaging was carried out
Bio-Imaging of Single Upconversion Nanoparticles
PLOS ONE | www.plosone.org 2 May 2013 | Volume 8 | Issue 5 | e63292
gradually zooming into areas of interest to track the UCNP
location precisely.
2.2.2 Laser-illuminated inverted epi-luminescence
microscope. A wide-field inverted epi-luminescence micro-
scope (Olympus IX70) equipped with a water-immersion objective
(406, NA 1.15, Olympus) was modified to allow external laser
illumination at the sample plane (fiber-coupled diode laser at
wavelength 978 nm, LD980-01CW CXCH-Photonics). In order
to achieve uniform illumination of the size-controllable field-of-
view at the sample plane, a modified Kohler illumination scheme
was designed and built in-house, as shown in Figure 1, with
detailed description and ray diagram provided in Figure S1. In
brief, an image of the field diaphragm placed in the excitation
beam path was formed at the sample plane (conjugate planes FD
and SP, Figure 1), while the excitation beam was homogenized
and collimated (owing to the conjugate planes LS and BFP,
Figure 1). In order to reduce laser speckle induced sample
illumination non-uniformity, the laser-coupling fiber was mechan-
ically dithered at a frequency .5 KHz. A zero-aperture adjustable
iris diaphragm served as the field diaphragm allowing tailoring of
the field-of-view at the sample plane, to a diameter as small as
20 mm that contained only several UCNPs. The sample image was
captured by an electron-multiplying CCD (EMCCD) camera
(Andor iXon DU-885) mounted to the microscope detection port.
An acousto-optic tunable filter (AOTF) (LSi-300 Hyperspectral
Imaging System, Gooch and Housego) was integrated into the
detection path of the epi-luminescence microscope for hyperspec-
tral imaging. The employed spectral range was 500–700 nm with
3-nm increments and a bandwidth of 3.7 nm. The spectral filter
module (filter cube) contained a high-pass absorbance filter (cut-off
850 nm, Thorlabs) placed in the excitation beam path to remove
the 978-nm laser side lobs. A dichroic beam-splitter (cut-off,
511 nm, Semrock) reflected the excitation light toward the sample,
while passing the emitted light to the detection path. Two
additional short-pass filters (cut-off, 842 nm, Semrock and cut-off,
700 nm, Thorlabs) in the detection channel suppressed the
excitation light leakage (see Figure S2). At the sample plane, the
TEM finder grid with UCNPs was placed on a microscope glass
slide and covered with a standard cover slip.
2.3 Emission Spectra (Ensemble)The UCNP powder was placed in a custom-designed sample
holder that consisted of a thin glass plate (thickness, 0.45 mm) with
a 1.5-mm diameter circular hole in the center sandwiched between
two glass cover-slips. The excitation fiber was butted against the
glass facing the hole filled with the UCNP powder. The emission
spectrum was measured in transmission using a calibrated fiber-
coupled diffraction-grating based spectrometer (Ocean Optics) in
conjunction with a short-pass emission filter (cut-off, 842 nm).
2.4 Conversion Efficiency2.4.1 Absolute conversion efficiency of UCNPs
(ensemble). The UCNP powder was placed in the custom-
designed sample holder (see section 2.3) at one of the exit ports of a
4-inch integrating sphere (Labsphere), as described by Page et al
[15]. At the backside of the sample holder, a tilted aluminum-
coated glass slide was placed to reflect the light back into the
integrating sphere avoiding a double-pass through the sample. A
multimode optical illumination fiber (fiber core 400 mm, NA 0.22)
was butted against the sample holder at the center of the hole. This
assured precise control and reproducibility of the excitation spot
size at the UCNP sample to calculate Iex. Luminescence emitted
by the UCNP sample was spatially integrated in the sphere by
means of multiple reflections from the walls, and eventually
detected using a photodiode (PD) (Thorlabs, PDA-55) at the exit
port positioned at 90u with respect to the illumination path. Using
a lock-in amplifier (Stanford Research Systems, model SR830)
connected to the PD output and pulsed laser excitation (laser:
LD980-01CW CXCH-Photonics; and pulse generator: Stanford
research systems, model DG535, pulse width 4 ms, frequency
125 Hz) enabled reliable registration of the UCNP luminescence
signals as low as 1 mW. The Iex range was controlled within several
decades by means of the laser diode current and neutral density
filters. The UCNP emission and unabsorbed excitation powers
were measured by inserting a short-pass (cut-off, 842 nm) and
long-pass (cut-off, 830 nm, Semrock) filter, respectively, in the
detection channel (in front of the PD). The performance of the
interference filters and the detector channel collection efficiency
were optimized by careful positioning a high-power lens in the
detection channel. The spectral response of the integrating sphere
and PD was calibrated for a broad spectral range (470–1050 nm).
By definition, guc was calculated as Pem/Pabs [W/W]. Pem was
determined by the straightforward measurement of the lumines-
cence signal corrected for the absolute spectral response of the
system and the UCNP spectral emission (see Figure S3A). Pabs was
determined by measuring the excitation powers unabsorbed by a
reference sample (TiO2), Pref, and the UCNP powder, PUCNP, so
that Pabs = Pref – PUCNP. Pabs mainly resulted from the linear
absorption of Ytterbion ions. For robust estimation of Pabs, the
measurements were repeated for several excitation intensities and
a linear fit was performed to calculate the absorbed fraction of the
excitation light (r2.0.99).
2.4.2. Absolute conversion efficiency of single-
UCNP. UCNPs sparsely deposited on the TEM finder grid
were imaged using the epi-luminescence microscope set-up, as
described earlier. The emitted power was determined by reading
out the pixel values of the EMCCD image using the camera
settings, sensitivity specifications and by calibrating the throughput
of the microscope optics from sample plane to image plane. Two
aspects of the EMCCD related to the epi-luminescence imaging
are important to note: 1. The sensor quantum efficiency,
QECCD(l) [e- per photon] is wavelength-dependent, so that
integration over the relevant wavelength range (400–800 nm)
was necessary. 2. The camera electron-multiplication gain (EM
gain) allowed straightforward multiplication of the pixel read-out
values by the EM gain (in virtue of RealGainTM EMCCD feature).
The UCNP signal read out by the sensor, SUCNP, depended on the
UCNP sample emitted intensity and the spectral calibration
coefficient of the imaging system according to:
SUCNP~NYBsYbabsIexgucftotalzNel , ð1Þ
where SUCNP and Nel [counts/s] were the signal per UCNP node
and electronic noise level, respectively; NYB was the number of
Ytterbium (Yb3+) ions per node that absorbed the excitation light;
sabs[cm2] was the Yb3+ absorption cross-section (neglecting the
absorption due to ,10-fold fewer Er3+ ions and sErabsvvsYb
abs)
[15,26]; and ftotal [counts/W/s] was the calibration coefficient
integrated over the UCNP emission spectrum (see Figure S4).
SUCNP was the sum of the pixels that sampled an image of the
UCNP node. The noise contained electronic dark and read noise
components. Optical background was negligible, as shown in
Figure S2. The number of Yb3+ per UCNP node was estimated
using the NaYF4 crystal lattice constants (a = 5.991 A, c = 3.526 A)
[27], TEM-derived dimensions and numbers, and Na to Yb molar
ratio [28].
Bio-Imaging of Single Upconversion Nanoparticles
PLOS ONE | www.plosone.org 3 May 2013 | Volume 8 | Issue 5 | e63292
ftotal was calculated according to:
ftotal~
ðl~800
l~400
QECCD(l)joptics(l)
hv(l)Nph(l)dl
EMgain:T
SCCD
ð2Þ
where joptics is the throughput of the imaging optics, h is Planck’s
constant, n the light frequency, Nph the number of photons per
wavelength per UCNP emission power, T the exposure time [s],
EMgain is a linear factor and SCCD is the camera sensitivity [e- per
count]. The values of QECCD and SCCD were provided by the
EMCCD manufacturer. The absolute throughput joptics was
spectrally calibrated by imaging of an optical fiber in the sample
plane and comparing the known fiber output power to the power
detected by the camera at the relevant wavelength range (see
Figure S5). In SI (Figure S4), the spectral output and the detector
response are shown for UCNP imaging using a water- and blood-
immersion objective.
Results and Discussion
Realization of the prime goal of ultrahigh-sensitivity imaging of
UCNPs in biological tissue is critically dependent on the attainable
UCNP contrast, which is defined as the ratio of the detected
luminescence signal originating from the UCNPs (S) to the
background signal stemming from the residual biological tissue
autofluorescence and noise (B). The signal estimation calls for a
thorough characterization of the excitation and emission proper-
ties of UCNPs, as well as quantification of the excitation/detection
paths of the optical microscopy system adapted for ultrahigh-
sensitivity imaging, where the adverse effects of the biological
tissue on UCNP excitation and detection are taken into account.
These effects are greatly exacerbated in the case of most
fluorescent organic dyes, as will be shown in our cross-comparison
study of optical imaging of a UCNP and fluorescein dye molecule
in biological tissue. The background signal estimation demands
consideration of the tissue optical properties, including scattering,
absorption and autofluorescence. We commence by reporting on
the measurement of absolute conversion efficiency and emission
spectra of UCNPs, using an integrating sphere set-up (Section
3.1.2; ensemble measurements) and calibrated optical microscopy
system (Section 3.1.3 and 3.1.4; single-particle measurements). In
Section 3.2 the quantitative assessment of the single-UCNP
imaging contrast in biological environment is addressed experi-
mentally using hemolzyed blood and theoretically using a skin
model. We combine the physical characteristics of UCNPs, the
optical properties of biological tissue and the abilities of advanced
imaging systems to provide clear guidance towards intelligent
development and a realistic application scope of upconversion
nanomaterials.
Figure 1. Diagram of the custom-modified epi-luminescence imaging system employed for single-UCNP and spectral imaging. Awide-field inverted epi-luminescence microscope was modified to allow external fiber-coupled laser illumination. The optical fiber was dithered toaverage out speckles. The excitation light was configured to uniformly illuminate the field-of-view at the sample plane via a modified Kohlerillumination scheme. The sample plane was imaged using an EMCCD camera, optionally mounted with an AOTF for hyperspectral imaging. Anadjustable iris diaphragm allowed reduction of the field-of-view to restrict imaging to several single UCNP particles and small clusters.doi:10.1371/journal.pone.0063292.g001
Bio-Imaging of Single Upconversion Nanoparticles
PLOS ONE | www.plosone.org 4 May 2013 | Volume 8 | Issue 5 | e63292
3.1 Quantitative Characterization3.1.1 Conversion efficiency. Comments on
conventions. Conversion efficiency, guc of UCNPs represents
the most important parameter that governs their luminescence
properties. It is defined as emitted power/absorbed power (Pem/
Pabs) expressed in W/W [15]. We note that this important
parameter is used inconsistently across the literature. Tradition-
ally, the term ‘‘quantum yield’’ (QY) is defined as the ratio of the
number of photons emitted to the number of photons absorbed by
the sample [29]. However, since during the upconversion process
one photon is emitted as a result of the absorption of two or more
photons, the QY would not exceed 50% following this definition.
In order to re-normalize this value, the QY of 2- or 3- photon
absorption processes is scaled by a factor 2 or 3 respectively
[16,30], although not consistently in literature [13,31]. In addition,
the emission is result of a complex, unknown combination of 2-
and 3-photon excitation pathways [32,33], rendering this
approach speculative. For these reasons, the term ‘‘conversion
efficiency’’ is believed to more adequately represent the net output
of the complex upconversion process.
It is worthwhile to comment here on two other issues with
UCNP conventions. Firstly, the dependency of guc on Iex
deteriorates the comparison of guc or QY measurements taken
at different values of Iex. For example, the reported QY values of
0.18% [22] and 0.47% [13] measured at Iex , 103 W/cm2 and
17.5 W/cm2 respectively, are hardly comparable due to an
unknown functional dependence of guc on Iex. Secondly,
quantitative reports on the absolute guc or related values are still
scarce in literature [15,31] often replaced by the limited-accuracy
comparisons of UCNP emission with that of bulk phosphors or
organic dyes [6,23]. In order to address this lacuna, we present the
results of the measurements of absolute guc of UCNPs synthesized
in-house for a large range of excitation intensities up to Isat.
3.1.2 Photophysical properties of as-synthesized
upconversion nanoparticles. We carried out characterization
of the key photophysical properties of as-synthesized UCNPs in
powder form. The b-NaYF4 nanocrystals were synthesized
following the thermal decomposition method, and co-doped with
Yb3+ and Er3+ ions (b-NaYF4:Yb,Er) at the most efficient molar
ratio of 20% Yb3+ and 2% Er3+ [5,28,32]. The measurement of
the absolute conversion efficiency versus excitation intensity at an
excitation wavelength (lex) of 978 nm, was performed using a
custom-modified integrating sphere set up. The integrating sphere
ensured absolute measurement of the absorption and emission
characteristics of UCNP powder independent of scattering by the
sample. Size, morphology and guc results are summarized in
Figure 2 and full scale TEM images at different magnification
levels are provided in Figure S6. The synthesis yielded quasi-
spherical UCNPs of diameters measured to be 68616 nm. The
guc was measured to reach nearly 2% at Iex ; Isat > 150 W/cm2, a
high value in comparison with that of the comparably sized b-
NaYF4:Yb,Er sample reported by Boyer et al. [31], (QY < 0.3%
at comparable Iex, recalculated using the emission spectrum and
photon energies to guc < 0.5%), although the sample environment
(organic solvent hexane known to quench guc) might well account
for this difference. As was expected for the supralinear upconver-
sion process, the guc dropped dramatically at low Iex, as can be
seen in Figure 2A. The UCNP emission spectra versus Iex were
acquired using a calibrated spectrometer and presented in Figure
S3. These spectra exhibited two characteristic emission bands
grouped in green (510–560 nm) and red (640–680 nm) emission
multiplets, as shown in Figure S3A. The red and green emission
bands were attributed to the Er atomic transitions induced by the
sequential two and three photon energy absorption processes [10].
The complex multi-step excitation process of the UCNPs is
nonlinear, as manifested by the supralinear dependence of the
emitted luminescence power, Pem, versus Iex: Pem*Inex, where the
power index n varies versus Iex reaching n = 1 at Iex = Isat. At low
Iex, n would ideally take discrete values of 2 or 3, reflecting the 2 or
3- step excitation process. However, the measured n takes the
values of 1.5 and 1.9 for the green and red emission bands
respectively (Figure S3C), reflecting more complex processes such
as linear decay, exited state absorption, energy transfer upconver-
sion, cross-relaxation and quenching, and their interrelations in
the different upconversion pathways [16,28,33–35]. It is beyond
the scope of this paper to elucidate these mechanisms. However, it
is important to account for the spectral and guc dependence on the
excitation intensity in a quantitative analysis of the UCNP imaging
in vivo.
The complexity of the upconversion excitation/emission
process is confounded by ensemble averaging, where the surface-
related (non-radiative relaxation) processes can be concealed by
inter-particle interactions. Characterization of an isolated individ-
ual UCNP is instrumental to remove this uncertainty.
3.1.3 Single-particle spectral imaging. A single upconver-
sion nanoparticle represents an excellent entity for the quantitative
measurements of the emission spectra and guc, because it is
unaffected by inter-particle interactions and has known physical
dimensions. In order to establish this single-UCNP experimental
model, we modified a wide-field epi-luminescence inverted
microscope to allow uniform illumination of the sample plane
with an external excitation laser (978 nm). A high-sensitivity
EMCCD camera was incorporated in the microscope detection
path. This imaging modality conferred several advantages,
Figure 2. Conversion efficiency, size and morphology of UCNPssynthesized in-house. (A) Plot of the absolute conversion efficiency(guc) [W/W] of the reported upconversion nanoparticle sample versusthe excitation intensity at lex = 978 nm measured using a calibratedintegrating sphere set-up. guc is the ratio of the emitted powerintegrated over the entire emission spectral range (500–700 nm) to theabsorbed power. (B) Size histogram obtained by analyzing thetransmission electron microscopy (TEM) images of NaYF4:Yb,Er UCNPs(330 particles). A typical TEM-image is shown in (C).doi:10.1371/journal.pone.0063292.g002
Bio-Imaging of Single Upconversion Nanoparticles
PLOS ONE | www.plosone.org 5 May 2013 | Volume 8 | Issue 5 | e63292
including short acquisition times (,1 s) at moderate Iex > 250 W/
cm2 uniformly distributed across the field-of-view, as compared to
flying-spot configurations that operate at Iex >106 W/cm2 and
with a pixel dwell time of 10 ms [22,24], resulting in acquisition
times of 40 minutes for a 5126512 pixel image. In order to
demonstrate single-UCNP imaging, the as-synthesized powder was
dispersed in organic solvent and sparsely deposited on a TEM grid
pre-coated with a Formav� monolayer film. This enabled TEM
and epi-luminescence imaging of the same sample areas to be
matched and individual nanoparticles singled out. An example of
this correlative imaging method is shown in Figure 3, where the
two top panels display the matched constellation of UCNPs
acquired by TEM and epi-luminescence microscopy, respectively,
and a single particle (designated ‘‘single’’) is observable.
The utility of this single-UCNP imaging system is demonstrated
by comparison of the luminescence spectra of single, clustered, and
powder UCNP samples that were obtained using an acousto-optic
tunable filter (AOTF) integrated into the detection path of the epi-
luminescence microscope. This enabled acquisition of spectral
data from every pixel, converting our microscope into a
hyperspectral imaging modality. The spectral responses of ‘‘single’’
and ‘‘cluster 2’’ were examined individually and locally by reading
out spectral data from the corresponding pixels (Figures 4A, 3B).
The emission spectra of the other small clusters were similar (data
not shown). The ensemble-averaged spectrum of the UCNP
powder was also obtained using the hyperspectral imaging mode
(Figure 4C) by processing a full-field image of the powder sample.
In order to cross-validate the spectral measurements, the UCNP
powder emission spectra were additionally acquired with a
diffraction-grating based calibrated spectrometer (dashed curves,
Figure 4). The dependency of the spectral features on excitation
intensity is discussed in Section 3.1.2 and Figure S3. The spectral
emissions of the individual, clustered and ensemble UCNPs
showed a high resemblance at equivalent Iex. Hence, the UCNP
spectral luminescence profile appeared to be primarily dependent
on Iex and independent on the inter-particle interactions in air,
thus clarifying the speculations made by Wu et al. [24] This
enabled accurate characterization of guc of single UCNPs, as
described in Section 3.1.4.
3.1.4. Measurement of single-particle conversion
efficiency. Each node of the UCNP constellation shown in
Figure 3 can be characterized in terms of the number/dimension
of nanoparticles per node and corresponding photon detection
rate (encoded in false color in Figure 3B) that can be converted to
photon emission rate (Pem) per excitation intensity, which suffices
to determine the UCNP conversion efficiency, guc = Pem/Pabs. The
photon emission rate integrated over the node was obtained by
reading out and converting the corresponding image pixels, since
the epi-luminescence detection channel throughput, and the
EMCCD spectral quantum efficiency versus the camera settings
were calibrated (see Section 2.4.2, Figure S5 and Figure S4). The
conversion efficiency was found by:
guc~Pem
Pabs
~(SUCNP{Nel)=ftotal
NYBsabsIex
, ð3Þ
where the quantities are explained in section 2.4.2.
The calculated guc of the single UCNP and two clusters (as
identified in Figure 3) obtained by processing the image data is
presented in Table 1 along with the relevant parameters. The
excitation intensity corresponded to the saturation regime where
the ensemble-averaged guc measured with the integrating sphere
was nearly 2%. The calculated values of guc of the individual
UCNP constellation nodes ranged between 1.2 and 2.0%, due to
the variability in the host crystal composition and impurities. The
independent guc measurements for the isolated and ensemble
UCNPs exhibited excellent agreement, which ensured that the guc
dependence on Iex and emission peak ratios hold for single UCNPs.
Although the absolute guc of the single-UCNP was marginally
lower than that of the ensemble-averaged UCNPs, it was
adequately high to enable ultrasensitive imaging at the single-
particle sensitivity level in biological scenarios, as addressed in the
following section.
3.2 Biomedical Imaging Applications3.2.1 Single-particle imaging through hemolyzed
blood. The feasibility of single-UCNP imaging in a biological
environment was explored by obscuring the UCNP sample with a
highly absorbing biological fluid, such as blood, with results
presented in Figure 5. Whole blood (1 ml) was hemolyzed by
replacing plasma with distilled water. After centrifuging the
supernatant was used to replace the water between the objective
and sample plane to create a ‘‘blood-immersion objective’’, as
shown in Figure 5A. The blood layer thickness was ,250 mm.
Hemoglobin, the oxygen-carrying protein in red blood cells, is the
main absorber in blood and has a distinct absorption spectrum, as
shown in Figure 5B. The UCNP emission spectrum is superim-
posed on the blood absorption spectrum to emphasize the high
absorption in the green emission band and low absorption in the
red emission band. This differential absorption by the blood
altered the sample coloration, as shown in Figure 5C. A low-
magnification image of the UCNP sample illuminated by the 978-
nm laser was captured by a digital color camera through the
eyepiece port of the epi-luminescence microscope, which repro-
duced the color perception by the human eye. The UCNP sample
appeared green (Figure 5C, top panel), when using the standard
water-immersion configuration of the objective lens, despite the
prominent red band in the UCNP emission spectrum. This is
explained by the higher spectral sensitivity of the eye to green color
compared to that of red color (,20 times [36]), which was also
reproduced in the spectral sensitivity of the color camera. In case
of the hemolyzed ‘‘blood-immersion objective’’, however
(Figure 5C, bottom panel), the green emitted light was largely
absorbed by the hemoglobin, and the UCNP sample appeared red
in the digital color image and to the human eye. Hence, the
UCNP red emission and NIR excitation passed with small losses
through the biological fluid, thus suggesting the feasibility of
imaging single UCNPs. This ultrahigh-sensitivity imaging through
an obscuring biological fluid was demonstrated, as shown in
Figure 5D, where the previously identified single UCNP was
clearly observable. The single-UCNP imaging has been reported
using flying-spot [22,24] and wide-field [23,25] set ups, for
exposed particles. To the best of our knowledge, this is the first
demonstration of single-UCNP imaging in a biological environ-
ment, using a moderate excitation intensity (250 W/cm2)
approaching the laser safety limits in a biological sample of
realistic thickness. The demonstrated imaging through a 250-mm
thick hemolyzed blood layer is, for example, favorably comparable
with the diameters of blood vessels in the microcirculation, which
can be found in proximity to organ surfaces [37].
These experimental results show the feasibility of optical
imaging of single upconversion nanoparticles in biological fluids,
in accordance with the assessment of the photophysical properties.
These results constitute an experimental platform from which the
detection limits of single-particle imaging in live skin is assessed
theoretically, as presented in the next section.
3.2.2 Modeling of single-particle imaging in skin. In
comparison with conventional in vivo fluorescence-assisted imag-
Bio-Imaging of Single Upconversion Nanoparticles
PLOS ONE | www.plosone.org 6 May 2013 | Volume 8 | Issue 5 | e63292
ing, UCNP-assisted imaging offers the advantage of reduced
excitation/emission light losses and complete suppression of the
optical background due to tissue autofluorescence [11]. The
implication of this improvement is demonstrated by a simplified
quantitative comparison of imaging in skin using fluorescein (FL)
and UCNPs at the ultrahigh-sensitivity single-particle level. The
choice for this representative visible organic fluorescent dye is
dictated by its broad acceptance in the field of experimental skin
research in vivo in animals and humans [38], as well as in
diagnostic procedures in skin [39] and other organs [40]. In our
model, a single emitter (FL and UCNP) is considered buried in
skin at a depth z. The calculated single-emitter (S) and background
(B) signals were compared in terms of their contrast (S/B) at
different z. A confocal imaging setting is assumed in this model to
Figure 3. Single-UCNP correlative imaging. (A) TEM and (B) epi-luminescence microscopy images corresponding to the same areas of thesample TEM grid. The distances between the individual (encircled) nanoparticles/clusters, given in (A), were precisely matched to those in (B) toidentify the same UCNP constellation. (C) Close-up TEM image of the same area as in (A), where UCNP sites designated ‘cluster 1’, ‘cluster 2’, and‘single’ correspond to the three sites in (B). The individual UCNPs within ‘cluster 1’ and ‘cluster 2’ were optically unresolvable. ‘‘Single’’ designates asingle UCNP particle clearly observable, as a diffraction-limited spot in (B). The excitation wavelength, intensity and exposure time were 978 nm,,250 W/cm2 and 0.7 s, respectively. The pixel values were converted to photons/second (ph/s) and color-coded according to the look-up color bar in(B).doi:10.1371/journal.pone.0063292.g003
Bio-Imaging of Single Upconversion Nanoparticles
PLOS ONE | www.plosone.org 7 May 2013 | Volume 8 | Issue 5 | e63292
Figure 4. Spectral imaging of UCNPs. Emission spectra of UCNPs in (A) single, (B) small cluster (designated ‘cluster 2’ in Figure 2) and (C) powderform (data points joined by solid lines) captured using hyperspectral epi-luminescence microscopy, overlaid with the ensemble-averaged spectra of
Bio-Imaging of Single Upconversion Nanoparticles
PLOS ONE | www.plosone.org 8 May 2013 | Volume 8 | Issue 5 | e63292
reduce the adverse obscuring effect of out-of-focus signals that
burden fluorescence-assisted imaging. The confocal imaging is
known to improve the signal discrimination, such that the signals
primarily originated from the focal volume at z.
The excitation power, P0ex at the focal volume is attenuated
according to Beer-Lambert’s law:Pex(z)~P0exe{mtrz, where mtr is
the transport attenuation coefficient [mm21] defined as a sum of
the attenuation, ma and reduced scattering coefficient, ms’ = (1-g)ms,
ms being the scattering coefficient, and g – the anisotropy factor.
The attenuation is due to photon removal from the focusing path
by absorption (ma ) and isotropic scattering (ms’). mtr was computed
in the visible and NIR spectral range using the reported values of
ma and ms’ for fresh Caucasian adult skin samples in vitro; while
assuming a layered skin structure of epidermis, dermis and
subcutaneous fat of corresponding thicknesses 80 mm, 400 mm and
520 mm, respectively [41]. The emitted power was calculated
using PUCNPem ~NybsYb
absIexgucand PFLem~1:sFL
absIexqFL where Iex –
the excitation intensity at depth z calculated by dividing Pex(z) by
the excitation area. WFL – the FL conversion efficiency was taken
as 0.9 and sFLabs = 3.5610216 cm2 at lex = 500 nm, [7] and 1
signifies a single FL emitter. NYB%1:1|105was calculated using
the NaYF4 crystal lattice constants [27] and Na to Yb molar ratio
[28] for a 70 nm particle, guc was obtained using the data plotted
in Figure 2A at the corresponding values of Iex, and sYBabs as listed in
Table 1. The maximum permissible exposure (MPE) for skin is
tabulated as 200 W/cm2 at 500 nm (FL) and 700 W/cm2 at
978 nm (UCNP) for a 1-ms exposure time [42]. The UCNP
emission is attenuated along the return path through skin, which
was accounted for using mtr of skin at the UCNP and FL emission
wavelengths. The isotropic emission collection efficiency was
determined by the objective lens acceptance angle (NA = 0.8). The
photon conversion of the collected emission by the EMCCD
camera (moderate EM gain, 6100) was computed using the
calibration protocol presented earlier (Equation 3, Section 3.1.4.).
The single-UCNP imaging contrast was estimated using the
background level set by the electronic noise, with dark and read
noise components specified by the camera manufacturer (Andor),
and shot noise calculated asffiffiffiffiffiffiffiffiffiNe{
p(Ne{ , the photoelectron number
before the EM-gain). The total noise was multiplied by the EM-
characteristic multiplicative noise factorffiffiffi2p
. [43] The FL imaging
background (B), however, had an additional autofluorescence-
induced component. At lex = 500 nm, the autofluorescence was
mainly due to the abundant endogenous fluorophore in skin, flavin
adenine dinucleotide (FAD), whose concentration in skin was
reported to be ,1 mg/mg [44]. ,750 FAD molecules were
situated in a 1-mm3 focal volume and contributed to B. In addition,
the out-of-focus FAD-induced background signal was estimated
following the model of confocal imaging by Magnor et al. for
scattering media, assuming a homogeneous distribution of FAD in
skin [45]. sFADabs and WFAD were taken as 6.0610218 cm2 at 500 nm
and 0.033, respectively [46]. The FAD-induced electronic signal at
the EMCCD camera was computed analogously to that of FL and
UCNP, corrected for the spectral filtering of the FAD emission
profile extending beyond the FL emission spectrum.
Figure 6 shows a plot of the calculated imaging contrast of a
single emitter versus depth in skin tissue. The absolute lumines-
cence signal S from FL is high at the shallow imaging depths
(,300 mm) in comparison with that of UCNP, and so is B due to
the autofluorescence by FAD (Figure 6, inset). The resultant
contrast provided by FL is therefore very modest, with the highest
value of 2 at the skin surface. Taking into consideration the non-
uniformity of autofluorescence across the skin sample, single-FL
imaging is at best problematic, if at all possible, particularly given
the serious issue of the short lifetime of FL before undergoing
photobleaching. In regard to S/B of the single UCNP, the
relatively low S was offset by the extremely small B, almost
invariable versus depth. The contrast is therefore, much higher,
slowly decreasing from a value of 10 down to 3 over the 200-mm
depth, which allows reliable detection of a single UCNP particle.
Based on our model S/B reached a value of 1 at 400-mm depth in
skin, however, due to a number of the simplifying assumptions
made this is likely to be the best-case-scenario.
3.2.3 Feasibility of single-particle imaging in
biotissue. The remarkable progress in the synthesis of upcon-
version nanomaterials and demonstration of their unique lumi-
nescence properties have led to new possibilities in optical
biomedical imaging using UCNPs. However, an uptake of this
luminescent nanotechnology into cellular imaging is tempered by
the existence of competitive luminescent nanomaterials that
exhibit a high contrast against the dim autofluorescence
background of cells (e.g. europium complexes, quantum dots).
Nam et al. reported the imaging of UCNP-labeled live HeLa cells
for 6 h using 980 nm excitation at Iex = 3 kW/cm2, which allowed
high-sensitivity intracellular imaging [47]. At these excitation
intensities, in vitro single UCNP imaging in cells is feasible, as our
results suggest. The scope of in vivo UCNP applications for optical
deep tissue imaging of small lesions appears to be limited by the
supralinear dependence of the upconversion luminescence on the
excitation intensity, which is greatly diminished at Iex%Isat in deep
(.1 cm) tissue layers. This confines the application niche of
UCNP-based molecular probes to optical luminescent imaging in
biological fluids, subsurface tissue layers and thick biological tissue
slices. Within these domains, the UCNP-based molecular probes
exhibit outstanding performance and provide unparalleled imag-
ing capabilities, as was demonstrated and modeled in this paper
UCNP powder captured by a calibrated spectrometer (dashed lines). The corresponding exposure times and EMCCD camera electron-multiplication(EM) gains were (A) 4 sec and 255; (B) 1.5 sec and 44; and (C) 0.014 sec and 9. Since the samples (A) and (B) contained considerably less emitters thanthe powder sample (C), the excitation intensities at lex = 978 nm were varied, respectively, from 250 W/cm2 to 8 W/cm2 to accommodate for thelarge disparity in the emission signals that would otherwise exceed the dynamic range of the EMCCD. The decreased Iex resulted in an increasedgreen-to-red emission ratio in (C) due to the varied upconversion energy redistribution between the green and red multiplets. Top panel, schematicdiagram of NaYF4:Yb,Er UCNP.doi:10.1371/journal.pone.0063292.g004
Table 1. Determination of the absolute guc using data fromthe calibrated epi-luminescence imaging of single and smallclusters of UCNPs.
Pem [W](25%) NYb (10%) sabs [cm2] b)
Iex [W/cm2](10%) guc [%]
Cluster 1a) 2.6610214 5.56105 1.0610220 2.66102 2.060.5
Cluster 2a) 2.2610214 6.16105 1.0610220 2.66102 1.360.3
Singlea) 4.9610215 2.36105 1.0610220 2.66102 1.260.3
a)As identified in Figures 3B and 3C. b)The Yb3+ absorption cross-section, as inRefs 15, 26. Unites are given in square brackets, percentage standard deviationsare given in brackets, except for guc.doi:10.1371/journal.pone.0063292.t001
Bio-Imaging of Single Upconversion Nanoparticles
PLOS ONE | www.plosone.org 9 May 2013 | Volume 8 | Issue 5 | e63292
(Single-particle imaging, Section 3.2, Figures 5 and 6). Such
exceptional performance is achieved due to the unique photo-
physical properties of UCNP that allows evading the adverse
effects of high absorption and autofluorescence of biological tissue,
as long as the high excitation intensity is attainable by focusing.
The theoretical analysis highlights great promise for imaging
single nanoparticles embedded in biological tissue, and provides a
useful guidance to luminescent nanoparticle design (not necessarily
limited to UCNP) and optical imaging modality. For example, an
improved photochemical design of UCNPs, e.g. co-doping NaYF4
with Ytterbium and Thulium featuring an emission peak at
800 nm should further reduce the absorption by blood (Figure 5)
and result in an increased imaging depth [48]. A promising recent
result was the synthesis of 42-nm UCNPs with an exceptional high
conversion efficiency at 800 nm (3.5%) [49], using core-shell
synthesis to reduce surface quenching [50]. The maximum
imaging depth (SNR .1) for the reported UCNP sample, with
mean size 42 nm and the highest reported value of guc = 3.5%, was
found to be 450 mm (see Figure S7).
There are several reports demonstrating UCNP-assisted imag-
ing depths of 2 cm [13] and 3.2 cm [51] in live mice and fat tissue
respectively, using a large number of UCNPs. In this paper, the
absolute detection sensitivity of single UCNP imaging in vivo is
addressed, which can readily be extended to an ensemble of
UCNPs.
At the same time, our presented optical imaging model was
oversimplified. The confocal flying-spot scanning modality is
Figure 5. Epi-luminescence imaging of a single UCNP using a ‘‘blood-immersion’’ objective. (A) A photograph of the hemolyzed bloodlayer between the objective and cover slip. (B) Absorption spectrum of the hemolyzed blood (red solid curve) and UCNP emission spectrum (greendashed curve). (C) Low-magnification images of the UCNP sample recorded through the eyepiece port using the water- (top) and blood- (bottom)immersion objective. The dried UCNP colloid rims appeared green (top) and red (bottom) due to the green light absorption by blood. (D) Epi-luminescence microscopy image of the UCNP constellation identified in Figure 2C, imaged using the blood-immersion objective. The single UCNP isclearly observable, although blurred. The EMCCD camera settings and excitation parameters were equivalent to these of Figure 2. The pixel valueswere converted to photons/second (ph/s) and color-coded using the look-up bar in (D).doi:10.1371/journal.pone.0063292.g005
Figure 6. Theoretical estimation of single-emitter detectionsensitivity in skin. A plot of the optical (confocal) detection contrastsof a single upconversion nanoparticle (UCNP, brown) and organicfluorescence dye (fluorescein, FC, green) versus their depth in skin, asmodeled theoretically. The inset shows more detailed quantitative plotsof the imaging signal (dashed) and background (dotted) of UCNP andFL versus depth in skin expressed in electrons per second (e2/s). Theblack dotted line demarcates the contrast value of 1. See text for details.doi:10.1371/journal.pone.0063292.g006
Bio-Imaging of Single Upconversion Nanoparticles
PLOS ONE | www.plosone.org 10 May 2013 | Volume 8 | Issue 5 | e63292
impractically slow due the long (sub-milliseconds) UCNP lumi-
nescence lifetime, which requires to the dwell time to be set to
,500 ms per pixel to avoid smearing of the emission light onto
adjacent pixels due to the UCNP after-glow [21]. On the other
hand, the wide-field epi-luminescence modality suffers from the
overshadowing of small single-UCNP signals by large UCNP
clusters, so that on some occasions, we had to reduce the field
diaphragm of the epi-luminescence microscope to collapse the
field-of-view to a diameter of ,20 mm. The wide-field configura-
tion was not efficient in suppressing out-of-focus signals, which
precluded the use of whole blood due to scattering, and required
blood hemolyzation. This could possibly be prevented by optical
clearing mechanisms that result in a reduced attenuation
coefficient of whole blood [52]. Lastly, the NIR irradiation dose
(250 W/cm2 CW) still exceeded the maximum permissible
exposure for skin. Although excitation at 980 nm at a ten-fold
greater intensity has been shown to be tolerable to live cells for 6 h
in vitro [47], continuous in vivo imaging at the excitation intensity as
low as 0.5 W/cm2 caused significant skin damage to a mouse [53],
due to the high water absorption in the live animal. Shifting the
excitation wavelength from 980 to 915 nm was suggested to
reduce water absorption. A pulsed laser excitation regime
delivering high energy per pulse at a low repetition rate represents
another possible solution to maintain acceptable imaging contrast
within the laser safety limits. Therefore, advanced optical imaging
– possibly, a hybrid of the wide-field epi-luminescence and
confocal modalities – is required to harness the UCNP specificity
and uniqueness.
There are a number of interesting prospects to employ UCNPs
as molecular probes in tissues sites accessible to ballistic photons
including skin, the superficial microcirculation and sub-surface
lesions for luminescence-guided surgery. Recent reports demon-
strate that imaging of low amounts of UCNP-labeled stem cells is
feasible: as few as ,10 labeled cells were subcutaneously detected
in nude mice and in vivo detection limits for UCNP-based imaging
were an order of magnitude lower in comparison with QDs
[54,55]. UCNPs conjugated to a cancer-targeting peptide, for
example somatostatin [56,57] can facilitate the diagnosis of skin
cancer [58]; detection of circulating tumor cells in subcutaneous
blood vessels, or the sensitive guidance to tumor sites by receptor-
targeted optical imaging [59]. Recently, we have successfully
conjugated UCNPs with scFv4D5 mini-antibodies raised against
the human epidermal growth factor receptor (HER2/neu)
overexpressed in e.g. breast adenocarcinoma cells and demon-
strated high-specific binding in vitro (unpublished results). Delivery
of conjugated UCNPs to tumors in vivo poses new challenges. The
primary delivery mechanism of intravenous injected nanoparticles
is thought to be the enhanced permeability and retention effect,
caused by leaky blood vessel walls in tumor tissue. Subsequent
nanoparticle diffusion into the tumor core occurs via intercellular
or intracellular routes, where the latter mediated by endocytosis is
more likely than the former due to the large nanoparticle size
compared to the intercellular space [47,60].
As a last example, UCNP-facilitated ion sensing by combining
the optical properties of ion sensing chromophores/fluorophores
with UCNP optical characteristics seems to be promising [61].
ConclusionThe adoption of upconversion nanomaterials in optical
biomedical imaging demands specification of application areas
where the UCNP merits are critical while the limitations are
tolerable, and where the cutting-edge sensitivity required for
UCNP-assisted imaging performance is achievable. We examined
this ultrahigh-sensitivity optical imaging scenario by performing
imaging and characterization of a single UCNP in a biological
environment represented by hemolyzed blood. In particular, the
key UCNP parameters of absolute conversion efficiency and
spectral emission were measured in individual and ensemble
particles and found comparable. These experimental results were
utilized in an idealized theoretical model, which was believed to
aid identification of application areas of extreme sensitivity in
challenging in vivo environments, including biological liquids,
subsurface layers and thick tissue slices. Specifically, our theoret-
ical skin imaging model showed that UCNP imaging had superior
contrast over that of conventional fluorescent dyes. The
background-free imaging of single upconversion nanoparticles at
depths up to 400 mm in skin was found feasible. Therefore, the
application scope of carefully tailored UCNP-based molecular
probes is significant, including luminescence-guided surgery and
ultrahigh-sensitivity bioassays in unprocessed biological fluids.
Supporting InformationSupporting information is available online and includes
additional information regarding the quantitative imaging method
and characterization of the UCNPs.
Supporting Information
Figure S1 Set-up and Kohler ray diagram. The set-up
consisted of a wide-field inverted epi-fluorescence microscope
(Olympus IX70) equipped with a water-immersion objective (406,
NA 1.15, Olympus). The laser illumination was guided to the
sample plane using a modified illumination path replacing the
default Mercury/Xenon arc lamp housing. To achieve homoge-
neous illumination at the sample plane and to have precise control
over the field-of-view, a modified Kohler illumination scheme was
built as is shown in panel (a). The two Kohler illumination ray
diagrams illustrating the homogeneous illumination light ray path
and the image forming light ray path are shown in panels (k-1) and
(k-2) respectively. The conjugate planes are explained in the
legend. In (k-1) a collimated laser beam filled the adjustable iris
located at the back focal plane of the field lens, which was, in turn,
one focal distance away from the back focal plane of the objective
resulting in even and collimated sample illumination. In (k-2) the
image forming function of the Kohler scheme is depicted: the
image of the iris located at the focal point of the field lens resulted
in the formation of its image at the objective focal plane. The
magnification of the iris image was the ratio between the focal
lengths of the objective and the field lens (,286 in this setup). A
zero-aperture iris was used to control the illumination area. The
other components of the set-up are an electron-multiplying CCD
(EMCCD) camera (Andor iXon DU-885) and an acousto-optic
tunable filter (AOTF) (LSi-300 Hyperspectral Imaging System,
Gooch and Housego) mounted to the left side port of the
microscope; excitation and emission filters; a 978 nm laser
(LD980-01CW, CXCH-Photonics) and an mechanical dither to
average out speckles. These components are further discussed in
the main paper.
(TIF)
Figure S2 Determination of background excitationlight. To estimate the amount of excitation light bleeding through
the emission filter set and reaching the EMCCD camera, we
imaged a TEM-grid with reference material (TiO2) and obtained
the pixel values on a line through the field-of-view on the image.
The graph shows the pixel values in counts for each pixel along the
line (pixel number) for the reference TEM-grid and for a TEM-
grid with UCNP sample deposited. As can be seen no elevation of
pixel value was observed for the reference image at the field-of-
Bio-Imaging of Single Upconversion Nanoparticles
PLOS ONE | www.plosone.org 11 May 2013 | Volume 8 | Issue 5 | e63292
view, whereas the sample image pixel values increased at the focus
spot due to detection of upconverted light. The detected signal was
nonzero due to addition of a pixel base level and (low) values for
read and dark noise. We can conclude that virtually no excitation
light reached the detector.
(TIF)
Figure S3 Excitation intensity dependence of the greenand red emission bands. (A) Ensemble emission spectra of
UCNP powder were measured in transmission using a customized
glass sample holder and calibrated spectrometer for several
excitation intensities at excitation wavelength 978 nm (see section
2.3). The spectra were normalized at the 540 nm peak and are
presented with an offset for clarity. (B) Integrating the spectra of
(A) over the green and red emission band resulted in a green-to-
red-ratio (GRR) dependence on excitation intensity. In (C), the
emitted power dependence on excitation intensity in the green and
red emission band is plotted on a double-logarithmic scale that
showed a linear slope until emission saturation is reached around
150 W/cm2. The slope is 1.9 for red emission and 1.5 for green
emission, confirming a nonlinear emission process that involves at
least 2 photons. Panel (D) shows the conversion efficiency (guc)
dependence of the green and the red emission bands on excitation
intensity.
(TIF)
Figure S4 Calibrated detector response for singleUCNP immersed in water or blood. Using the emission
spectrum of the single UCNP (Figure 4A) and Equation 2 the
spectral calibration coefficient in counts/W/s for each wavelength
can be calculated assuming T = 1 s, EM gain = 1, Sccd = 0.89
e2/count and Nph the number of photons per wavelength per
UCNP emission power of 1 Watt. Data points are connected by a
solid line. In case of the hemolyzed blood-immersion objective the
spectral detector response is reduced due to absorption of light by
hemoglobin. The absorption is high for the green emission band
and low for the red emission band. Integrating over all
wavelengths results in total counts/W/s for UCNP emission,
referred to as ftotal.
(TIF)
Figure S5 Spectral throughput of inverted epi-lumines-cence microscopy system. The absolute throughput, joptics,
defined as signalsensor-plane/signalsample-plane, of the microscope imaging
system was obtained by imaging an optical fiber (multimode,
100 mm core, NA 0.22) placed at the sample plane of the
microscope. The output power of the fiber was measured using a
calibrated power meter (Thorlabs). Using the camera settings and
sensitivity specifications the power detected by the sensor can be
calculated from the image pixel values, and subsequently the
throughput can be obtained. This was done for several
wavelengths and spectrally interpolated using a white light source
(Ocean Optics LS-1, tungsten halogen), as plotted in the graph.
The discontinuity around 510 nm was due to the dichroic beam
splitter with a cut-off wavelength at 511 nm. Assuming isotropic
emission of upconverted light by the UCNPs, the actual
throughput of the system needed to be corrected for the
acceptance angle of the microscope objective, a factor 1/3 for
NA = 1.15 (not shown).
(TIF)
Figure S6 TEM images with different magnifications ofin-house synthesized UCNPs.(TIF)
Figure S7 Theoretical estimation of single UCNP detec-tion sensitivity in skin, a comparison between in-housesynthesized and state-of-the-art UCNPs. The highest
conversion efficiency of 3.5% was reported for Yb,Tm-co-doped
core-shell UCNPs (ref 49). Using our model described in Section
3.2.2, we calculated the imaging signal contrast and plotted the
result (blue dot-dashed line) together with the previous results for
fluorescein and the 70-nm UCNP. The 42-nm 3.5%-UCNP has a
reduced absorption of excitation light due to the smaller size of the
particle which reduced the imaging contrast, but the higher guc
and the reduced transport attenuation coefficient of skin for the
luminescence signal resulted in a higher contrast at increasing
depth. The maximum imaging depth in skin is 450 mm, with
contrast 1.
(TIF)
Acknowledgments
This work was performed in part at the OptoFab node of the Australian
National Fabrication Facility, a company established under the National
Collaborative Research Infrastructure Strategy to provide nano and
microfabrication facilities for Australia’s researchers.
Author Contributions
Conceived and designed the experiments: AN VKAS ZS EAG VAS VYP
AVZ. Performed the experiments: AN VKAS ZS EAG AVN VAS.
Analyzed the data: AN VKAS EAG AVZ. Contributed reagents/
materials/analysis tools: AN VKAS ZS EAG AVN VAS. Wrote the
paper: AN VKAS ZS AVN VAS VYP AVZ.
References
1. Hilderbrand SA, Weissleder R (2009) Near-Infrared Fluorescence: Application
to In Vivo Molecular Imaging. Curr Opin Chem Biol 14: 9–71.
2. Patterson MS, Chance B, Wilson BC (1989) Time Resolved Reflectance and
Transmittance for the Non-Invasive Measurement of Tissue Optical Properties.
Appl Opt 28: 2331–2336.
3. Corlu A, Choe R, Durduran T, Rosen MA, Schweiger M, et al. (2007) Three-
Dimensional In Vivo Fluorescence Diffuse Optical Tomography of Breast Cancer
in Humans. Opt Express 15: 6696–6716.
4. Schaafsma BE, Mieog JSD, Hutteman M, van der Vorst JR, Kuppen PJK, et al.
(2011) The clinical use of indocyanine green as a near-infrared fluorescent
contrast agent for image-guided oncologic surgery. J Surg Oncol 104: 323–332.
5. Heer S, Kompe K, Gudel HU, Haase M (2004) Highly Efficient Multicolour
Upconversion Emission in Transparent Colloids of Lanthanide-Doped NaYF4
Nanocrystals. Adv Mater 16: 2102–2105.
6. Yi G, Lu H, Zhao S, Ge Y, Yang W, et al. (2004) Synthesis, Characterization,
and Biological Application of Size-controlled Nanocrystalline NaYF4: Yb, Er
Infrared-to-Visible Up-Conversion Phosphors. Nano Lett 4: 2191–2196.
7. Resch-Genger U, Grabolle M, Cavaliere-Jaricot S, Nitschke R, Nann T (2008)
Quantum Dots Versus Organic Dyes as Fluorescent Labels. Nat Methods 5:
763–775.
8. Mancini MC, Kairdolf BA, Smith AM, Nie S (2008) Oxidative Quenching and
Degradation of Polymer-encapsulated Quantum Dots: New Insights Into the
Long-Term Fate and Toxicity of Nanocrystals In Vivo. J Am Chem Soc 130:
10836–10837.
9. Liu K, Liu X, Zeng Q, Zhang Y, Tu L, et al. (2012) Covalently Assembled NIR
Nanoplatform for Simultaneous Fluorescence Imaging and Photodynamic
Therapy of Cancer Cells. ACS nano 6: 4054–4062.
10. Auzel F (2004) Upconversion and Anti-Stokes Processes With f and d Ions in
Solids. Chem Rev 104: 139–174.
11. Xu CT, Svensson N, Axelsson J, Svenmarker P, Somesfalean G, et al. (2008)
Autofluorescence Insensitive Imaging Using Upconverting Nanocrystals in
Scattering Media. Appl Phys Lett 93: 171103.
12. Leblond F, Davis SC, Valdes PA, Pogue BW (2010) Pre-Clinical Whole-Body
Fluorescence Imaging: Review of Instruments, Methods and Applications.
J Photochem Photobiol, B 98: 77–94.
13. Liu Q, Sun Y, Yang T, Feng W, Li C, et al. (2011) Sub-10 nm Hexagonal
Lanthanide-Doped NaLuF4 Upconversion Nanocrystals for Sensitive Bioima-
ging in Vivo. J Am Chem Soc 133: 17122–17125.
14. Vinegoni C, Razansky D, Hilderbrand SA, Shao F, Ntziachristos V, et al. (2009)
Transillumination Fluorescence Imaging in Mice Using Biocompatible Upcon-
verting Nanoparticles. Opt Lett 34: 2566–2568.
Bio-Imaging of Single Upconversion Nanoparticles
PLOS ONE | www.plosone.org 12 May 2013 | Volume 8 | Issue 5 | e63292
15. Page RH, Schaffers KI, Waide PA, Tassano JB, Payne SA, et al. (1998)
Upconversion-pumped Luminescence Efficiency of Rare-Earth-doped Hosts
Sensitized With Trivalent Ytterbium. JOSA B 15: 996–1008.
16. Suyver J, Aebischer A, Biner D, Gerner P, Grimm J, et al. (2005) Novel
Materials Doped With Trivalent Lanthanides and Transition Metal Ions
Showing Near-Infrared to Visible Photon Upconversion. Opt Mater 27: 1111–
1130.
17. Hehlen MP, Frei G, Gudel HU (1994) Dynamics of infrared-to-visible
upconversion in Cs3Lu2Br9: 1% Er3+. Phys Rev B 50: 16264.
18. Zipfel WR, Williams RM, Christie R, Nikitin AY, Hyman BT, et al. (2003) Live
Tissue Intrinsic Emission Microscopy Using Multiphoton-excited Native
Fluorescence and Second Harmonic Generation. Proc Natl Acad Sci U S A
100: 7075.
19. Wang F, Wang J, Liu X (2010) Direct Evidence of a Surface Quenching Effect
on Size-Dependent Luminescence of Upconversion Nanoparticles. Angew
Chem Int Ed 122: 7618–7622.
20. Kelf T, Sreenivasan V, Sun J, Kim E, Goldys E, et al. (2010) Non-Specific
Cellular Uptake of Surface-Functionalized Quantum Dots. Nanotechnology 21:
285105.
21. Pichaandi J, Boyer JC, Delaney KR, van Veggel FCJM (2011) Two-Photon
Upconversion Laser (Scanning and Wide Field) Microscopy using Ln3+-Doped
NaYF4 Upconverting Nanocrystals A Critical Evaluation of their Performance
and Potential in Bio-imaging. J Phys Chem C: 19054–19064.
22. Ostrowski AD, Chan EM, Gargas DJ, Katz EM, Han G, et al. (2012) Controlled
Synthesis and Single Particle Imaging of Bright, Sub-10 nm Lanthanide-Doped
Upconverting Nanocrystals. ACS nano 6: 2686–2692.
23. Mialon G, Turkcan S, Dantelle G, Collins DP, Hadjipanayi M, et al. (2010)
High Up-Conversion Efficiency of YVO4: Yb, Er Nanoparticles in Water Down
to the Single-Particle Level. J Phys Chem C: 22449–22454.
24. Wu S, Han G, Milliron DJ, Aloni S, Altoe V, et al. (2009) Non-blinking and
Photostable Upconverted Luminescence from Single Lanthanide-Doped Nano-
crystals. Proc Natl Acad Sci U S A 106: 10917–10921.
25. Park YI, Kim JH, Lee KT, Jeon KS, Na HB, et al. (2009) Nonblinking and
nonbleaching upconverting nanoparticles as an optical imaging nanoprobe and
T1 magnetic resonance imaging contrast agent. Adv Mater 21: 4467–4471.
26. Lakshminarayana G, Ruan J, Qiu J (2009) NIR Luminescence From Er-Yb, Bi-
Yb and Bi-Nd Codoped Germanate Glasses for Optical Amplification. J Alloys
Compd 476: 878–883.
27. Mai HX, Zhang YW, Si R, Yan ZG, Sun L, et al. (2006) High-Quality sodium
rare-earth fluoride nanocrystals: controlled synthesis and optical properties. J Am
Chem Soc 128: 6426–6436.
28. Kramer KW, Biner D, Frei G, Gudel HU, Hehlen MP, et al. (2004) Hexagonal
Sodium Yttrium Fluoride Based Green and Blue Emitting Upconversion
Phosphors. Chem Mater 16: 1244–1251.
29. Melhuish W, Zander M (1984) Nomenclature, Symbols, Units and Their Usage
in Spectrochemical Analysis-VII. Molecular Absorption Spectroscopy, Ultravi-
olet and Visible (UV/VIS) Pure Appl Chem 56: 231–245.
30. Tikhomirov V, Adamo G, Nikolaenko A, Rodriguez V, Gredin P, et al. (2010)
Cathodo-and photoluminescence in Yb3+ Er3+ Co-Doped PbF2 Nanoparticles.
Opt Express 18: 8836–8846.
31. Boyer JC, Van Veggel FCJM (2010) Absolute Quantum Yield Measurements of
Colloidal NaYF4: Er3+, Yb3+ Upconverting Nanoparticles. Nanoscale 2: 1417–
1419.
32. Mai HX, Zhang YW, Sun LD, Yan CH (2007) Highly Efficient Multicolor Up-
conversion Emissions and Their Mechanisms of Monodisperse NaYF4: Yb, Er
Core and Core/Shell-Structured Nanocrystals. J Phys Chem C 111: 13721–
13729.
33. Wang Y, Tu L, Zhao J, Sun Y, Kong X, et al. (2009) Upconversion
Luminescence of b-NaYF4: Yb3+, Er3+@ b-NaYF4 Core/Shell Nanoparticles:
Excitation Power Density and Surface Dependence. J Phys Chem C 113: 7164–
7169.
34. Pollnau M, Gamelin D, Luthi S, Gudel H, Hehlen M (2000) Power Dependence
of Upconversion Luminescence in Lanthanide and Transition-Metal-Ion
Systems. Phys Rev B 61: 3337.
35. Shan J, Uddi M, Wei R, Yao N, Ju Y (2010) The Hidden Effects of Particle
Shape and Criteria for Evaluating the Upconversion Luminescence of the
Lanthanide Doped Nanophosphors. J Phys Chem C 114: 2452–2461.
36. (1931) Commission Internationale de l’Eclairage Proceedings. Cambridge,
United Kingdom: Cambridge University Press.
37. Boerma EC, Mathura K, van der Voort P, Spronk P, Ince C (2005) Quantifying
Bedside-derived Imaging of Microcirculatory Abnormalities in Septic Patients: A
Prospective Validation Study. Crit Care 9: R601–R606.
38. Bollinger A, Frey J, Jager K, Furrer J, Seglias J, et al. (1982) Patterns of Diffusion
Through Skin Capillaries in Patients With Long-Term Diabetes. N Engl J Med307: 1305–1310.
39. Dietterle S, Lademann J, Rowert-Huber HJ, Stockfleth E, Antoniou C, et al.
(2008) In-Vivo Diagnosis and Non-Inasive Monitoring of Imiquimod 5% Creamfor Non-Melanoma Skin Cancer Using Confocal Laser Scanning Microscopy.
Laser Phys Lett 5: 752–759.40. Hsiung PL, Hardy J, Friedland S, Soetikno R, Du CB, et al. (2008) Detection of
colonic dysplasia in vivo using a targeted heptapeptide and confocal micro-
endoscopy. Nat Med 14: 454–458.41. Salomatina E, Jiang B, Novak J, Yaroslavsky AN (2006) Optical properties of
normal and cancerous human skin in the visible and near-infrared spectralrange. J Biomed Opt 11: 064026.
42. (2012) Standards Australia Limited/Standards New Zealand, AS/NZS 60825.Sydney, AU and Wellington, NZ: SAI Global Limited. 1–48 p.
43. Robbins MS, Hadwen BJ (2003) The noise performance of electron multiplying
charge-coupled devices. IEEE Transactions on Electronic Devices 50: 1227–1232.
44. Bessey OA, Lowry OH, Love RH (1949) The Fluorimetric Measurement of theNucleotides or Riboflavind and Their Concentration in Tissues. J Biol Chem
180: 755–769.
45. Magnor M, Dorn P, Rudolph W (2001) Simulation of confocal microscopythrough scattering media with and without time gating. JOSA B 18: 1695–1700.
46. Islam SDM, Susdorf T, Penzkofer A, Hegemann P (2003) Fluorescencequenching of flavin adenine dinucleotide in aqueous solution by pH dependent
isomerisation and photo-induced electron transfer. Chem Phys 295: 137–149.47. Nam SH, Bae YM, Park YI, Kim JH, Kim HM, et al. (2011) Long-Term Real-
Time Tracking of Lanthanide Ion Doped Upconverting Nanoparticles in Living
Cells. Angew Chem 123: 6217–6221.48. Dong N, Pedroni M, Piccinelli F, Conti G, Sbarbati A, et al. (2011) NIR-to-NIR
Two-Photon Excited CaF2: Tm3+, Yb3+ Nanoparticles: Multifunctional Nanop-robes for Highly Penetrating Fluorescence Bio-Imaging. ACS nano 5: 8665–
8671.
49. Xu CT, Svenmarker P, Liu H, Wu X, Messing ME, et al. (2012) High-Resolution Fluorescence Diffuse Optical Tomography Developed with Nonlin-
ear Upconverting Nanoparticles. ACS nano: 4788–4795.50. Yi GS, Chow GM (2007) Water-soluble NaYF4: Yb, Er (Tm)/NaYF4/polymer
core/shell/shell nanoparticles with significant enhancement of upconversionfluorescence. Chem Mater 19: 341–343.
51. Chen G, Shen J, Ohulchanskyy TY, Patel NJ, Kutikov A, et al. (2012) (a-
NaYbF4: Tm3+)/CaF2 Core/Shell Nanoparticles with Efficient Near-Infrared toNear-Infrared Upconversion for High-Contrast Deep Tissue Bioimaging. ACS
nano: 8280–8287.52. Tuchin VV, Xu X, Wang RK (2002) Dynamic optical coherence tomography in
studies of optical clearing, sedimentation, and aggregation of immersed blood.
Appl Opt 41: 258–271.53. Zhan Q, Qian J, Liang H, Somesfalean G, Wang D, et al. (2011) Using 915-nm
Laser Excited Tm3+/Er3+/Ho3+ Doped NaYbF4 Upconversion Nanoparticlesfor In Vitro and Deeper In Vivo Bioimaging Without Overheating Irradiation.
ACS nano: 3744–3757.54. Wang C, Cheng L, Xu H, Liu Z (2012) Towards whole-body imaging at the
single cell level using ultra-sensitive stem cell labeling with oligo-arginine
modified upconversion nanoparticles. Biomaterials.55. Cheng L, Yang K, Zhang S, Shao M, Lee S, et al. (2010) Highly-sensitive
multiplexed in vivo imaging using PEGylated upconversion nanoparticles. NanoResearch 3: 722–732.
56. Sreenivasan VKA, Kim EJ, Goodchild AK, Connor M, Zvyagin AV (2012)
Targeting somatostatin receptors using in situ-bioconjugated fluorescentnanoparticles. Nanomedicine in press.
57. Sreenivasan VKA, Stremovskiy OA, Kelf TA, Heblinski M, Goodchild AK, etal. (2011) Pharmacological Characterization of a Recombinant, Fluorescent
Somatostatin Receptor Agonist. Bioconjugate Chem 22: 1768–1775.
58. Nguyen BD, McCullough AE (2002) Imaging of Merkel Cell Carcinoma1.Radiographics 22: 367–376.
59. Becker A, Hessenius C, Licha K, Ebert B, Sukowski U, et al. (2001) Receptor-targeted Optical Imaging of Tumors With Near-Infrared Fluorescent Ligands.
Nat Biotechnol 19: 327–331.60. Kim HR, Andrieux K, Gil S, Taverna M, Chacun H, et al. (2007) Translocation
of poly (ethylene glycol-co-hexadecyl) cyanoacrylate nanoparticles into rat brain
endothelial cells: role of apolipoproteins in receptor-mediated endocytosis.Biomacromolecules 8: 793–799.
61. Xie L, Qin Y, Chen H-Y (2013) Direct Fluorescent Measurement of BloodPotassium with Polymeric Optical Sensors Based on Upconverting Nanomater-
ials. Anal Chem. In press.
Bio-Imaging of Single Upconversion Nanoparticles
PLOS ONE | www.plosone.org 13 May 2013 | Volume 8 | Issue 5 | e63292
UlrichswebUlrich's Serials Analysis System
You are logged into UlrichsWeb
Quick Search
Advanced SearchBrowseListsHelpMy Account Ulrich's AlertUlrich's Update
P L o S One BACK TO RESULTS
Click highlighted text for a new search on that item.
ISSN: 1932-6203Title: P L o S One Additional Title Information
Publishing Body: Public Library of ScienceCountry: United StatesStatus: ActiveStart Year: 2006Frequency: IrregularDocument Type: Journal; Academic/ScholarlyRefereed: YesAbstracted/Indexed: YesMedia: Online - full textLanguage: Text in EnglishPrice: FreeSubject: SCIENCES: COMPREHENSIVE WORKS
MEDICAL SCIENCESDewey #: 500, 610LC#: Q179.9Editor(s): Damian PattinsonPublisher(s): Peter BinfieldURL: http://www.plosone.org/home.actionDescription: Covers primary research from all disciplines within science and medicine.
ADDITIONAL TITLE INFORMATION
Acronym Description: Public Library of ScienceAlternate Title: Variant format: PLoS ONE
Back to Top
Add this item to: Request this title: Print Download E-mail I'd like to request this title.
Corrections:
Submit corrections to Ulrich's about this title.
Publisher of this title?
If yes, click GO! to contact Ulrich's about updating your title listings in the Ulrich's database.
Back to Top
Copyright © 2010 ProQuest LLC | Privacy Policy | Terms of Use | Contact Us
Ulrichsweb.com--Full Citation http://www.ulrichsweb.com/ulrichsweb/Search/fullCitation.asp?navPa...
1 of 1 13/08/2010 3:16 PM